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1. Introduction and rationaleThe increasing incidence in urinary incontinence (UI)with advancing age is directly correlated to spontaneousapoptosis of the muscle cells of the rhabdosphincter, thestriated urethral sphincter. As life expectancy of the wholesociety is growing, significance of UI as a disease of theelderly will increase further. Age-dependent reduction in thenumber of striated muscle cells is now held the highestranking cause of urinary stress incontinence in elderlywomen and men (Strasser et al., 1999, 2000a,b).The technical availability of autologous satellite cellsobtained from skeletal muscle enabled the immediateapplication of muscle progenitor cells in the treatment ofUI by means of tissue engineering techniques in patients ofany age, including elderly (Strasser et al., 2004).This review focuses on the theoretical assumptions andexperimental findings on muscle derived stem cells (MDSC)justifying their straightforward application in urology. Onlyquite recently, the regenerative function of mammaliantissues has become understood as ubiquitous (Deans andMoseley, 2000; Moore, 2002). Not only rapidly renewingtissues like skin, bone marrow or blood were found topossess a stem cell reserve: even apparently quiescenttissues, like nerve tissue or heart muscle disclosed—duringcellular stress or tissue loss—a tissue inherent renewingpotential (Hanashima et al., 2004). Tissue renewal dependson the persistence of so-called adult stem cells or precursorsof mature cells.Moreover, not only acute processes like injury orintoxication activate stem cell directed tissue regeneration.Cellular aging, cell death (apoptosis and necrosis) andrenewal continue throughout life and through all tissues.Imbalances between cell death and renewal are now thoughtto occur during aging (when apoptosis supersedes renewingcapacity) and during malignant transformation (whenregular cell death is hindered by apoptosis inhibition)(Hanahan and Weinberg, 2000).According to this rationale, the higher prevalence ofUI among elderly persons of both sexes can beinterpreted as a symptom of increasingly poor andeventually failing tissue regeneration in the vesico-urethral apparatus.A direct correlation between age and the number ofapoptotic cells in the rhabdomyosphincter on the one hand,and impaired detrusor contractility and weak closurepressure caused by structural deficits of the rhabdosphincteron the other hand, suggest that stress incontinence ispredominantly associated with a dysfunction of the striatedurethral sphincter (Frauscher et al., 1998, Strasser et al.,1999).Yiou and co-workers have shown that the adultrhabdosphincter in mice regenerates after an injury bymeans of intrinsic satellite cells (Yiou et al., 2003). Thisfinding is interesting per se, because rhabdosphincter andstriated skeletal muscles have a different embryologicalorigin. Muscle cells of the external sphincter arethought to arise via transdifferentiation of urethral smoothmuscle cells (Borirakchanyavat et al., 1997)—and thus

We have analysed the voltage-gated ion channels and fusion competence of skeletal muscle myoblasts labelled with green fluorescent protein (GFP) and the membrane dye PKH transplanted into the infarcted myocardium of syngenic rats. After cell transplantation the animals were killed and GFP(+)-PKH(+) myoblasts enzymatically isolated for subsequent studies of ionic currents through voltage-gated sodium, calcium and potassium channels. A down-regulation of all three types of ion channels after engraftment was observed. The fraction of cells with calcium (68%) and sodium channels (65%) declined to zero within 24 h and 1 week, respectively. Down-regulation of potassium currents (90% in control) occurred within 2 weeks to about 30%. Before injection myoblasts expressed predominantly transient outward potassium channels whereas after isolation from the myocardium exclusively rapid delayed rectifier channels. The currents recovered completely between 1 and 6 weeks under cell culture conditions. The down-regulation of ion channels and changes in potassium current kinetics suggest that the environment provided by infarcted myocardium affects expression of voltage-gated ion channels of skeletal myoblasts.

BACKGROUND The electrophysiological properties of myoblast cultures established from the human and porcine rhabdosphincter (RS) and porcine lower limb muscle (LLSKM) were studied to elucidate their potential for tissue engineering applications in the lower urinary tract. METHODS Muscle biopsies were collected from the prostatic part of the RS, the RS of male pigs, and the porcine LLSKM. Ion channels were studied by means of the patch-clamp technique. RESULTS Only one subtype each of voltage gated Na+ and Ca2+ channels was observed in porcine RS and LLSKM. Two types of voltage gated Ca2+ channels were identified in human RS cells. The porcine RS and LLSKM myoblasts displayed similar fusion competence. CONCLUSIONS Porcine RS and LLSKM myoblasts and human RS and human skeletal muscle cells show a high degree of similarity. Injection of autologous skeletal muscle myoboblasts in the lower urinary tract might, therefore, represent a promising approach to treat stress incontinence after radical prostatectomy. © 2004 Wiley-Liss, Inc.

Objective Transurethral ultrasound-guided injection of autologous myoblasts has recently been shown to cure urinary stress incontinence. In the present study, the dose-dependent changes in maximal urethral closure pressures after application of myoblasts were investigated in a porcine animal model. Methods Myoblast cultures were grown from a porcine muscle biopsy. The biopsy was enzymatically dissociated by using a modified cell dispersion technique. Single myoblasts in suspension were manually collected with a micropipette under microscopic control. Next a clonal myoblast culture was prepared. Before the cells were applied, fluorescence labelling (PKH) was used to assess integration of the injected myoblasts into the rhabdosphincter. With the help of a transurethral ultrasound probe (23 F, 11MHz) and a special injection system, the myoblasts were injected into the rhabdosphincter of five pigs under direct sonographic control. Into two different areas of the rhabdosphincter, increasing different cell counts were injected (total volume 1.5ml). At each area, 10 depots of 150μl volume were injected all along the rhabdosphincter. The following cell counts were used: 1.5 × 10 6 , 2.1 × 10 6 , 4.2 × 10 6 (low range) 5.69 × 10 6 , 8.1 × 10 6 , 1.13 × 10 7 , 1.6 × 10 7 (mid range) 2.26 × 10 7 , 4.4 × 10 7 , and 7.8 × 10 7 (high range). To avoid possible cell rejection, we immunosuppressed the pigs with daily cortisone (1g Solu Dacortin) because allogenic myoblasts were used. Urethral pressure profiles (UPPs) were measured before and 3 wk postoperatively before the pigs were put to sleep. The lower urinary tract was removed in all pigs for histological analysis. Results Histological examination of the specimens revealed that the injected cells had survived at the injection site and had formed new myofibres. Overall the UPP curves revealed dose-dependent changes. Statistically significant increased pressure values of up to more than 300% could be observed in all cases in which higher concentrations of cells had been applied. Increases were also noted in mid range concentrations although not to such a high extent (approximately 150%). Pressure values had even diminished (approximately 50%) after injecting the three lowest concentrations (1.5 × 10 6 , 2.1 × 10 6 , 4.2 × 10 6 ). Conclusions The present results show that the effects after application of myoblasts into the rhabdosphincter are dose-dependent.

Defects caused by traumatic or postsurgical loss of muscle mass may result in severe impairments of the functionality of skeletal muscle. Tissue engineering represents a possible approach to replace the lost or defective muscle. The aim of this study was to compare the suitability of three different biomaterials as scaffolds for rat myoblasts, using a new animal model. PKH26-fluorescent-stained cultured rat myoblasts were either seeded onto polyglycolic acid meshes or, alternatively, suspended in alginate or in hyaluronic acid-hydrogels. In each of the eight Fisher CDF-344 rats, four capsule pouches were induced by subcutaneous implantation of four silicone sheets. After two weeks the silicone sheets were removed and myoblast-biomaterial-constructs were implanted in the preformed capsules. Specimens were harvested after four weeks and examined histologically by H&E-staining and fluorescence microscopy. All capsules were well-vascularized. Implanted myoblasts fused by forming multinucleated myotubes. This study demonstrates that myoblasts seeded onto different biomaterials can be successfully transplanted into preformed highly vascularized capsule pouches. Our animal model has paved the way for studies of myoblast-biomaterial transplantations into an ectopic non-muscular environment.

Objectives: To prove whether intramyocardial transplantation of combined skeletal myoblasts (SM) and mononuclear bone marrow stem cells is superior to the isolated transplantation of these cell types after myocardial infarction in rats. Methods: In 67 male Fischer rats myocardial infarction was induced by direct ligature of the LAD. Seven days postinfarction baseline echocardiography and intramyocardial cell transplantation were performed. Via lateral thoracotomy 200 ml containing either 10 7 SMs or 10 7 bone marrow-derived mononuclear cells (BM-MNC) or a combination of 5 £ 10 6 of both cell types (MB) were injected in 10 –15 sites in and around the infarct zone. In controls (C) 200 ml of cell-free medium were injected in the same manner. Before injection both cell types were stained using a fluorescent cell linker kit (PKH, Sigma). In addition, SMs were transfected with green fluorescent protein. Nine weeks postinfarction follow-up echocardiography was performed and animals were sacrificed for further analysis. Results: At baseline echocardiography there was no difference in left ventricular ejection fraction (LVEF; C, SM, BM-MNC, MB: 60.1 ^ 3.2, 53.3 ^ 10.2, 53.1 ^ 8.7, 49 ^ 9.0%) and left ventricular end diastolic diameter (LVEDD; C, SM, BM-MNC, MB: 6.5 ^ 0.8, 5.17 ^ 0.8, 5.77 ^ 1.4, 6.25 ^ 0.8 mm) between the different therapeutic groups. Eight weeks after cell transplantation LVEDD was significantly increased in all animals except those that received a combination of myoblasts and bone marrow stem cells (MB; C, SM, BM-MNC, MB: 7.7 ^ 0.6 mm, P ¼ 0:001; 7.7 ^ 1.5 mm, P , 0:001; 7.7 ^ 1.1 mm, P ¼ 0:005; 6.6 ^ 1.7 mm, P ¼ 0:397). At the same time LVEF decreased significantly in the control group (C), stayed unchanged in animals that received bone marrow stem cells (BM-MNC) and increased in animals that received myoblasts (SM) and a combination of both cell types (MB; C, SM, BM-MNC, MB: 45.3 ^ 7.0%, P ¼ 0:05; 63.9 ^ 15.4%, P ¼ 0:044; 54.3 ^ 6.3%, P ¼ 0:607; 63.0 ^ 11.5%, P ¼ 0:039). Conclusions: The present data show that the concept of combining SMs with bone marrow-derived stem cells may be of clinical relevance by merging the beneficial effects of each cell line and potentially reducing the required cell quantity. Further studies are required to identify the exact mechanisms underlying this synergy and to allow full exploitation of its therapeutic potential. q 2004 Elsevier B.V. All rights reserved.

Purpose Myoblast transplantation has been shown to augment damaged tissue. Moreover it could be used as an autologous bulking agent in refluxing vesicoureteral junctions in order to restore the damaged muscular integrity. But graft viability and the behaviour of the donor myoblasts at the ureterovesical junction have not been demonstrated yet. Material and methods Muscle biopsies from the back muscles of 5 pigs were obtained. Quality of the cells was evaluated by electrophysiological and immunohistochemical tests. Additionally they were labelled with a PKH fluorescent membrane dye to provide fluorescent marking of live cells. 5 weeks after muscle biopsies all animals underwent cell transplantation with 300 × 106 cells suspended in transplantation medium loaded into five 1-cc tuberculin syringes and injected into the ureterovesical junction. Animals were sacrificed 8 weeks later, the donor cells were identified using PKH26 labelling and fluorescence-activated cell-sorter analysis. Moreover histopathology and evaluation concerning tissue reaction at the donor site was also carried out. Results 2-3 intact autologous skeletal myoblast–derived skeletal muscle cells/visual field were found in all injected animals. However, immunohistochemistry demonstrated viability of transplanted myoblasts only at the borders of the bulking area wheras central widespread necrosis with red fluorescent cell detritus could be observed. We did not observe marked signs of an inflammatory response, even though a fibrotic scar was found at the site of injection. Conclusions These results highlight the inability of the vast majority of transplanted myoblasts to survive. Only a small portion of the cells survive when transplanted into a well vascularized area which is in favour of the assumption of an indirect impairment such as paracrine effects. Failure of myoblast transplantation in this animal study does not prove that the procedure itself is ineffective, but these results do caution against the application of in vivo myoblasts as a bulking agent.